Effect of neurturin deficiency on cholinergic and catecholaminergic innervation of the murine eye.

Neurturin (NRTN) is a neurotrophic factor required for the development of many parasympathetic neurons and normal cholinergic innervation of the heart, lacrimal glands and numerous other tissues. Previous studies with transgenic mouse models showed that NRTN is also essential for normal development and function of the retina (J. Neurosci. 28:4123-4135, 2008). NRTN knockout (KO) mice exhibit a marked thinning of the outer plexiform layer (OPL) of the retina, with reduced abundance of horizontal cell dendrites and axons, and aberrant projections of horizontal cells and bipolar cells into the outer nuclear layer. The effects of NRTN deletion on specific neurotransmitter systems in the retina and on cholinergic innervation of the iris are unknown. To begin addressing this deficiency, we used immunohistochemical methods to study cholinergic and noradrenergic innervation of the iris and the presence and localization of cholinergic and dopaminergic neurons and nerve fibers in eyes from adult male wild-type (WT) and NRTN KO mice (age 4-6 months). Mice were euthanized, and eyes were removed and fixed in cold neutral buffered formalin or 4% paraformaldehyde. Formalin-fixed eyes were embedded in paraffin, and 5 µm cross-sections were collected. Representative sections were stained with hematoxylin and eosin or processed for fluorescence immunohistochemistry after treatment for antigen retrieval. Whole mount preparations were dissected from paraformaldehyde fixed eyes and used for immunohistochemistry. Cholinergic and catecholaminergic nerve fibers were labeled with primary antibodies to the vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), respectively. Cholinergic and dopaminergic cell bodies were labeled with antibodies to choline acetyltransferase (ChAT) and TH, respectively. Cholinergic innervation of the mouse iris was restricted to the sphincter region, and noradrenergic fibers occurred throughout the iris and in the ciliary processes. This pattern was unaffected by deletion of NRTN. Furthermore, functional experiments demonstrated that cholinergic regulation of the pupil diameter was retained in NRTN KO mice. Hematoxylin and eosin stains of the retina confirmed a marked thinning of the OPL in KO mice. VAChT and ChAT staining of the retina revealed two bands of cholinergic processes in the inner plexiform layer, and these were unaffected by NRTN deletion. Likewise, NRTN deletion did not affect the abundance of ChAT-positive ganglion and amacrine cells. In marked contrast, staining for TH showed an increased abundance of dopaminergic processes in the OPL of retina from KO mice. Staining of retinal whole mounts for TH showed no difference in the abundance of dopaminergic amacrine cells between WT and KO mice. These findings demonstrate that the neurotrophic factor NRTN is not required for the development or maintenance of cholinergic innervation of the iris, cholinergic control of pupil diameter, or for development of cholinergic and dopaminergic amacrine cells of the retina. However, NRTN deficiency causes a marked reduction in the size of the OPL and aberrant growth of dopaminergic processes into this region.

[Characteristics of the autonomic nervous system state in children with myopia associated with undifferentiated connective tissue dysplasia].

Autonomic nervous system disorders occur in 100% cases in patients with connective tissue dysplasia. Biomechanism of accommodation and autonomic innervation of ciliary body was not investigated clearly and presented as area of further research. The goal of the study was to evaluate features of autonomic nervous system in patients with myopia associated with connective tissue dysplasia. 50 children with myopia associated with connective tissue dysplasia went through ophtholmological examination. To evaluate function of autonomic nervous system all patients were observed with the machine "Valenta" which has ECG recording. As a result we found that children with myopia and connective tissue dysplasia have unsatisfactory adaptive reserves with imbalance of sympathetic and parasympathetic input, autonomic nervous system wasn't available to provide homeostasis in demanding level. We hypothesized that autonomic nervous system might be a reason which lead to disorders in process of accommodation during myopia and assist its progressing.

Adenine nucleotide effect on intraocular pressure: Involvement of the parasympathetic nervous system.

Nucleotides are present in the aqueous humor possibly exerting physiological effects on intraocular pressure (IOP). To determine the effect of nucleotides such as ATP and its related derivatives on IOP, New Zealand white rabbits were used. IOP was measured in rabbits treated topically either with saline (control) or with a single dose (10 microg/microL) of adenine nucleotides (ATP, 2-meS-ATP, ATP-gamma-S, alpha,beta-meADP, alpha,beta-meATP and beta,gamma-meATP). Those nucleotides reducing IOP (alpha,beta-meATP and beta,gamma-meATP) were then tested in concentrations ranging from 1 to 100 microg/microL to obtain the IC(50) value. Several antagonists for the P2 and adenosine A1 receptors (all at 10 microg/microL) were assayed 30 min before the application of the hypotensive nucleotide beta,gamma-meATP. To see whether the nucleotide was acting directly on the structures involved in aqueous humor dynamics or on the autonomic nerves controlling IOP, animal denervation and sympathetic (yohimbine and ICI-118,551 at 10 microg/microL) and parasympathetic (atropine and hexametonium at 10 microg/microL) receptors' antagonists were used 30 min before the instillation of beta,gamma-meATP. alpha,beta-meATP and beta,gamma-meATP decreased IOP to 60% of control value (basal IOP=23.2+/-1.3 mmHg), with IC(50) of 1.59+/-0.21 microg/microLand 0.56+/-0.62 microg/microL, which corresponds to 3mM and 1mM respectively. Denervation completely abolished the effect of beta,gamma-meATP. Sympathetic antagonists did not modify the hypotensive effect of beta,gamma-meATP, but parasympathetic antagonists were able to abolish it. Among the series of adenine nucleotide tested, alpha,beta-meATP and beta,gamma-meATP presented hypotensive actions on IOP. beta,gamma-meATP seems to stimulate cholinergic terminals being its final effect the IOP reduction. Therefore, these two nucleotides are interesting pharmacological tools for those pathologies related with high intraocular pressure.

Changes in the number of chick ciliary ganglion neuron processes with time in cell culture.

The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.

The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture.

Acetylcholine receptors accumulate along the length of cholinergic neuron-skeletal muscle contacts in vitro. The main purpose of this study was to describe, in a quantitative way, the distribution of acetylcholine receptor clusters induced by ciliary ganglion neurons over a period of time extending from hours to weeks after contacts are established. Neurites were filled with Lucifer Yellow and receptor clusters were identified with rhodamine-bungarotoxin. A cluster located within 5 micron of a nerve process or 10 micron of the base of a growth cone was considered to be a neurite-associated receptor patch (NARP). The first synaptic potentials were evoked 20 min after growth cone-myotube contact, and, after 24 h of co-culture, greater than 60% of the nerve-muscle pairs tested were functionally connected. NARPs appear rapidly; the first clusters were detected approximately 6 h after the neurons were plated. They were composed of several small subclusters or speckles of rhodamine-bungarotoxin fluorescence. The initial accumulation of receptors may occur at the advancing tips of nerve processes because NARPs were found at greater than 80% of the growth cone-muscle contacts examined between 12 and 24 h of co-culture. Over the 3-wk period examined, the mean incidence of NARPs ranged between 1.0 and 2.6 per 100 micron of neurite-myotube contact, with the peak observed on the second day of co-culture. During the first 3 d in culture, when the neurons were multipolar, nearly all of the primary processes induced one or more clusters. With time, as the neurons become unipolar (Role and Fischbach, 1987) NARPs persisted along the remaining dominant process. Measurements made during the third day of co-culture suggest that NARPs disappear along shorter neurites before they retract. Synaptic currents were detected by focal extracellular recording at 55% of the NARPs. The fact that spontaneous or evoked responses were not recorded at 45% suggests that contacts with clusters exhibit two functional states. Two types of presynaptic specialization at identified NARPs observed by scanning electron microscopy appear to be correlated with the functional state.

Cholinergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons.

Chick ciliary ganglionic neurons require an interaction with their peripheral targets for survival during a critical period of their embryonic development in vivo. It has recently been shown that survival of these neurons in dissociated cell cultures is supported by extract from whole chick embryo. In this study, an assay system based on microwell cultures of ciliary ganglionic neurons was used to demonstrate that a very rich source of trophic factor for them is the intraocular target tissues they innervate. Out of 8000 trophic units present in a 12-day embryo, 2500 were contained in the eye. A subdissection of the eye showed its activity to be localized in a fraction containing the ciliary body and choroid coat, with a specific activity almost 20-fold higher than that of the whole embryo. This selective intraocular distribution at a time when survival or death of ciliary ganglionic neurons is decided in vivo suggests that this soluble factor may be involved in the normal development of the ciliary ganglion.

Adrenergic innervation of the parasympathetic ciliary ganglion in the chick.

The chick ciliary ganglion receives a nonvascular symathetic innervation in addition to the well-known cholinergic one; fluorescent, varicose adrenergic fibers form pericelluar baskets. Adrenergic fibes were identified electron microscopically in ganglia fixed with potassium permanganate. The fibers degenerate after injection of 6-hydroxydopamine. No true synaptic relationships involving adrenergic varicosities and ganglion cells or cholinergic terminals were demonstrable. The distribution of the adrenergic fibers suggests a kind of "distance à synapse" with the choroidal cells or with the preganglionic fibers (or both). The adrenergic innervation might provide a modulation of the cholinergic transmission.

Axotomy mimicked by localized colchicine application.

Comparable depression of synaptic transmission in the avian ciliary ganglion resulted from either section or localized colchicine treatment of the ciliary nerves. Both colchicine treatment and axotomny produced similar changes in RNA distribution in the cell bodies as well. Colchicine did not directly affect transmission, and action potential propagation along the ciliary nerves was normal. Interference with axoplasmic transport of material in both cases is postulated to signal the observed chromatolytic changes.

Effect of hydrogen sulfide on sympathetic neurotransmission and catecholamine levels in isolated porcine iris-ciliary body.

In the present study, we investigated the pharmacological action of hydrogen sulfide (H2S, using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na2S as donors) on sympathetic neurotransmission from isolated, superfused porcine iris-ciliary bodies. We also examined the effect of H2S on norepinephrine (NE), dopamine and epinephrine concentrations in isolated porcine anterior uvea. Release of [3H]NE was triggered by electrical field stimulation and basal catecholamine concentrations was measured by high performance liquid chromatography (HPLC). Both NaHS and Na2S caused a concentration-dependent inhibition of electrically evoked [3H]NE release from porcine iris-ciliary body without affecting basal [3H]NE efflux. The inhibitory action of H2S donors on NE release was attenuated by aminooxyacetic acid (AOA) and propargyglycine (PAG), inhibitors of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), respectively. With the exception of dopamine, NaHS caused a concentration-dependent reduction in endogenous NE and epinephrine concentrations in isolated iris-ciliary bodies. We conclude that H2S can inhibit sympathetic neurotransmission from isolated porcine anterior uvea, an effect that is dependent, at least in part, on intramural biosynthesis of this gas. Furthermore, the observed action of H2S donors on sympathetic transmission may be due to a direct action of this gas on neurotransmitter pools.

Pupil constriction evoked in vitro by stimulation of the oculomotor nerve in the turtle (Trachemys scripta elegans).

The pond turtle (Trachemys scripta elegans) exhibits a notably sluggish pupillary light reflex (PLR), with pupil constriction developing over several minutes following light onset. In the present study, we examined the dynamics of the efferent branch of the reflex in vitro using preparations consisting of either the isolated head or the enucleated eye. Stimulation of the oculomotor nerve (nIII) using 100-Hz current trains resulted in a maximal pupil constriction of 17.4% compared to 27.1% observed in the intact animal in response to light. When current amplitude was systematically increased from 1 to 400 microA, mean response latency decreased from 64 to 45 ms, but this change was not statistically significant. Hill equations fitted to these responses indicated a current threshold of 3.8 microA. Stimulation using single pulses evoked a smaller constriction (3.8%) with response latencies and threshold similar to that obtained using train stimulation. The response evoked by postganglionic stimulation of the ciliary nerve using 100-Hz trains was largely indistinguishable from that of train stimulation of nIII. However, application of single-pulse stimulation postganglionically resulted in smaller pupil constriction at all current levels relative to that of nIII stimulation, suggesting that there is amplification of efferent drive at the ganglion. Time constants for constrictions ranged from 88 to 154 ms with relaxations occurring more slowly at 174-361 ms. These values for timing from in vitro are much faster than the time constant 1.66 min obtained for the light response in the intact animal. The rapid dynamics of pupil constriction observed here suggest that the slow PLR of the turtle observed in vivo is not due to limitations of the efferent pathway. Rather, the sluggish response probably results from photoreceptive mechanisms or central processing.

Diurnal fluctuations and developmental changes in ocular dimensions and optical aberrations in young chicks.

PURPOSE: To investigate further the emmetropization process in young chicks by studying the diurnal fluctuations and developmental changes in the ocular dimensions and optical aberrations, including refractive errors, of normal eyes and eyes that had the ciliary nerve sectioned (CNX). METHODS: The ocular dimensions and aberrations in both eyes of eight CNX (surgery on right eyes only) and eight normal chicks were measured with high-frequency A-scan ultrasonography and aberrometry, respectively, four times a day on five different days from posthatching day 13 to 35. A fixed pupil size of 2 mm was used to analyze aberration data. Repeated-measures ANOVA was applied to examine the effects of age, time of day, and surgery. RESULTS: Refractive errors and most higher-order aberrations decreased with development in both normal and CNX eyes. However, although normal eyes showed a positive shift in spherical aberration with age, changing from negative spherical aberration initially, CNX eyes consistently exhibited positive spherical aberration. Anterior chamber depth, lens thickness, vitreous chamber depth, and thus optical axial length all increased with development. Many of these ocular parameters also underwent diurnal changes, and mostly these dynamic characteristics showed no age dependency and no effect of CNX. Anterior chamber depth, vitreous chamber depth, and optical axial length were all greater in the evening than in the morning, whereas the choroids were thinner in the evening. Paradoxically, eyes were more hyperopic in the evening, when they were longest. Although CNX eyes, having enlarged pupils, were exposed to larger higher-order aberrations, their growth pattern was similar to that of normal eyes. CONCLUSIONS: Young chicks that are still emmetropizing, show significant diurnal fluctuations in ocular dimensions and some optical aberrations, superimposed on overall increases in the former and developmental decreases in the latter, even when accommodation is prevented. The possibility that these diurnal fluctuations are used to decode the eye's refractive error status for emmetropization warrants investigation. That eyes undergoing ciliary nerve section have more higher-order aberrations but do not become myopic implies a threshold for retinal image degradation below which the emmetropization process is not affected.

Pulse-step models of control strategies for dynamic ocular accommodation and disaccommodation.

Dynamic properties and control strategies of step responses by accommodation and disaccommodation differ from one another. Peak velocity of accommodation increases with response magnitude, while peak velocity and peak acceleration of disaccommodation increase with starting position. These dynamic properties can be modeled as control strategies that use independent acceleration-pulse and velocity-step components that are integrated respectively into phasic-velocity signals that control movement and tonic-position signals that control magnitude. Accommodation is initiated toward its final destination by an acceleration-pulse whose width increases with response magnitude to increase peak velocity. Disaccommodation is initiated toward a default destination (the far point) by an acceleration-pulse whose height increases with dioptric distance of the starting position to increase peak velocity and peak acceleration. Both responses are completed and maintained by tonic-position signals whose amplitudes are proportional to the final destination. Mismatched amplitudes of phasic-velocity and tonic-position signals in disaccommodation produce unstable step responses.

Accommodation and presbyopia: the ciliary neuromuscular view.

Presbyopia (literally, "old eye"), the age-related loss of the ability to accommodate, is the most common ocular affliction in the world. Although the lens no doubt has a major role in presbyopia, altered lens function could be in part secondary to extralenticular age-related changes, such as loss of ciliary body forward movement. Centripetal ciliary muscle movement does not seem to decrease significantly with age. Loss of elasticity of the ciliary muscle posterior attachments may be an important factor contributing to presbyopia. Even if loss of ciliary muscle mobility is not causally related to presbyopia, it may limit the performance of putatively accommodating intraocular lenses now being developed by academic and industrial groups.

Anterior segment growth and peripheral neural pathways in chick.

PURPOSE: To learn if peripheral nerve pathways are necessary for corneal expansion and anterior segment growth under a 12-hr light:dark cycle or for the inhibition of corneal expansion under constant light rearing. METHODS: Recently hatched White Leghorn chicks under anesthesia received unilateral ciliary ganglionectomy (CGx), cranial cervical ganglionectomy (Sx), or section of the ophthalmic nerve (TGx), along with sham-operated and/or never-operated control cohorts. Chicks were reared postoperatively under either a 12-hr light:dark cycle or under constant light. After 2 weeks and with the chicks under anesthesia, corneal radii of curvature and diameters were obtained with a photokeratoscope, refractometry and A-scan ultrasonography were performed, and the axial and equatorial dimensions of enucleated eyes were measured with digital calipers. Corneal areas were calculated from corneal curvatures and diameters. RESULTS: Despite the rich peripheral innervation to the eye, the selective denervations performed here exerted remarkably limited effects on corneal expansion and anterior segment development in chicks reared under either lighting condition. Ophthalmic nerve section did reverse in large part the inhibition of equatorial expansion of the vitreous chamber occurring under constant light rearing. CONCLUSIONS: The ciliary, sympathetic, or ophthalmic peripheral nerve pathways to the eye are not required either for corneal expansion and anterior segment development under a 12-hr light:dark cycle or for the inhibition of corneal expansion under constant light rearing. The ocular sensory innervation may be a means for regulating vitreous cavity shape.

Stimulus requirements for the decoding of myopic and hyperopic defocus under single and competing defocus conditions in the chicken.

PURPOSE: The bidirectional nature of emmetropization, as observed in young chicks, implies that eyes are able to distinguish between myopic and hyperopic focusing errors. In the current study the spatial frequency and contrast dependence of this process were investigated in an experimental paradigm that allowed strict control over both parameters of the retinal image. Also investigated was the influence of accommodation. METHODS: Defocusing stimuli were presented through lens-cone devices with attached targets. These devices were monocularly applied to 5-day-old chickens for 4 days. Defocus conditions included: (1) 7 D of myopic defocus, (2) 7 D of hyperopic defocus, and (3) a combination of the two. Two high contrast target designs, a spatially rich, striped Maltese cross (target 1) and a standard Maltese cross (target 2) were used, except in some experiments where target contrast or spatial frequency content was further manipulated. To test the role of accommodation, the treated eye of some chicks underwent ciliary nerve section before attachment of the device. Refractive error (RE) was measured by retinoscopy and axial ocular dimensions measured by A-scan ultrasonography, both in chicks under anesthesia. RESULTS: With imposed myopic defocus and high contrast, target 1 elicited significantly better compensation than did target 2. With imposed hyperopic defocus, both targets elicited near normal compensatory responses. Reducing image contrast to 32% for target 2 and to 16% for target 1 precluded compensation for myopic defocus, inducing myopia instead. The low-pass-filtered target also induced myopia, irrespective of the sign of imposed defocus. With competing defocus and intact accommodation, target 1 induced a transient hyperopic growth response, whereas myopia was consistently observed with target 2. When accommodation was rendered inactive, both targets induced myopia under these competitive conditions. CONCLUSIONS: Compensation to myopic defocus is critically dependent on the inclusion of middle to high spatial frequencies in the stimulus and has a spatial frequency-dependent threshold contrast requirement. With competing myopic and hyperopic defocus, the former transiently dominates the latter as a determinant of ocular growth, provided that the stimulus conditions include sufficient middle to high spatial frequency information and that accommodation cues are available.

A pulse-step model of accommodation dynamics in the aging eye.

We have developed a dynamic model of accommodation that combines independent phasic-velocity and tonic-position neural signals to control position, velocity and acceleration properties of accommodative step responses. Phasic and tonic signals were obtained from neural integration of a fixed-height acceleration-pulse and variable-height velocity-step respectively to control independent acceleration and velocity properties of the step response. Duration and amplitude of the acceleration-pulse are increased with age to compensate for age-related increases of visco-elastic properties of the lens to maintain youthful velocity. The model illustrates a neural control strategy that is similar to the classical neural control model of step changes by the saccadic and vergence systems.

Lenticular accommodation in relation to ametropia: the chick model.

Our goal was to determine whether experimentally induced ametropias have an effect on lenticular accommodation and spherical aberration. Form-deprivation myopia and hyperopia were induced in one eye of hatchling chicks by application of a translucent goggle and +15 D lens, respectively. After 7 days, eyes were enucleated and lenses were optically scanned prior to accommodation, during accommodation, and after accommodation. Accommodation was induced by electrical stimulation of the ciliary nerve. Lenticular focal lengths for form-deprived eyes were significantly shorter than for their controls and accommodation-associated changes in focal length were significantly smaller in myopic eyes compared to their controls. For eyes imposed with +15 D blur, focal lengths were longer than those for their controls and accommodative changes were greater. Spherical aberration of the lens increased with accommodation in both form-deprived and lens-treated birds, but induction of ametropia had no effect on lenticular spherical aberration in general. Nonmonotonicity from lenticular spherical aberration increased during accommodation but effects of refractive error were equivocal. The crystalline lens contributes to refractive error changes of the eye both in the case of myopia and hyperopia. These changes are likely attributable to global changes in the size and shape of the eye.

Contribution of cerebrovascular parasympathetic and sensory innervation to the short-term control of blood flow in rat cerebral cortex.

In two groups of normotensive rats anaesthetised with halothane, either the nasociliary nerve (NCN) or the NCN and parasympathetic (PS) fibres together (NCN-PS) were functionally blocked at the right ethmoidal foramen. Blocking was achieved reversibly and repeatedly using a cooling probe. Cortical regional CBF (rCBF) was measured bilaterally using laser-Doppler probes. In Group 1, bilateral common carotid occlusion (CCO) was applied for 1 min both with and without block. In Group 2, CCO was applied permanently followed by stages of controlled haemorrhagic hypotension to deepen the ischaemia and the block applied at each stage. In Group 1, during CCO, rCBF was unaffected by blocking NCN-PS. However, during the transient postocclusive hyperaemia, blocking NCN-PS, but not NCN alone, significantly increased right side rCBF. In Group 2 and in Group 1 in the absence of CCO (normotension), rCBF was unaffected by blocking either set of fibres. We conclude that neither NCN nor PS fibres contribute to the tonic level of rCBF or to its autoregulatory control, but PS fibres conduct impulses tending to resolve postischaemic hyperaemia. We suggest that a subpopulation of PS fibres containing neuropeptide Y is activated under conditions of supernormal rCBF.

Transneuronal retinal input to the primate Edinger-Westphal nucleus.

The Edinger-Westphal nucleus of the oculomotor nuclear complex provides preganglionic parasympathetic innervation to the pupil. We labelled its retinal input by transneuronal autoradiography after an eye injection of [3H]proline in the macaque monkey. The primary retinal projection to the pretectum terminated in the ipsilateral and contralateral olivary nuclei. These nuclei were intensely labelled and sharply delimited, with a mean diameter of 590 microm and a rostrocaudal length of 2.52 mm. The caudal half of the olivary nucleus on each side broke into multiple clumps of label. Fragments of label also surrounded each olivary nucleus. The exact pattern of pretectal labelling varied considerably among animals and even from side to side in the same animal. In 5 of 6 monkeys, label from the olivary nucleus reached the Edinger-Westphal nucleus transneuronally. In transverse sections, the Edinger-Westphal label appeared as a circular patch located on either side of the midbrain ventral to the cerebral aqueduct in the central gray matter. It averaged 230 microm in diameter and 610 microm in length. In Nissl-stained sections, the autoradiographic label corresponded to a distinct nucleus comprised of neurons that were smaller than neurons in nearby somatic subdivisions of the oculomotor complex. The mean area of Edinger-Westphal neurons was 295 microm2. Transneuronal retinal input to the Edinger-Westphal nucleus mediating pupillary constriction terminates in a single, well-defined cell group in the midbrain.

Innervation of rat iris by trigeminal and ciliary neurons expressing pChAT, a novel splice variant of choline acetyltransferase.

We have recently discovered a splice variant of choline acetyltransferase (ChAT) mRNA and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, the presence of pChAT in rat iris was examined by immunohistochemistry and Western blot using a pChAT antiserum, in combination with RT-PCR analysis and ChAT enzyme assay. For comparison, the conventional ChAT (cChAT) was studied in parallel. By pChAT immunohistochemistry, intense labeling was found to occur in nerve fibers of the iris and in neurons of the ciliary and trigeminal ganglia. Denervation studies, analyzed by semiquantitative morphometry, indicated that these iridial pChAT fibers originated about half from the ciliary ganglion and the other half from the trigeminal ganglion. The presence of pChAT protein in the iris and trigeminal ganglion was confirmed by Western blot. The expression of pChAT mRNA in the ciliary and trigeminal ganglia was proved by RT-PCR. Although cChAT protein and mRNA were detected in the ciliary ganglion, neither was detectable in the trigeminal ganglion. The contributions of the ciliary and trigeminal ganglia to the iridial ChAT enzyme activity were verified by the present ChAT assay. Here, we provide evidence that iridial pChAT nerves are composed of postganglionic parasympathetic efferents from the ciliary ganglion and, more interestingly, somatic sensory afferents of the trigeminal ophthalmic nerve.